Structure and expression of the D-amino-acid oxidase gene from the yeast Rhodosporidium toruloides.
D-Amino-acid oxidase (DAO2; EC 1.4.3.3) catalyses the oxidative deamination of D-amino acids to alpha-keto acids and ammonia. The purified DAO protein from Rhodosporidium toruloides was used to determine its amino acid sequence. Three internal peptide sequences, YCQYLARELQ, IAGIDDQAAEPIR and RCTMDSSDP, were obtained and used to synthesize four fully degenerated oligonucleotides for cloning of the DAO gene. Both cDNA and genomic DNA encoding R. toruloides DAO were cloned and sequenced. Comparison of these two DNA sequences revealed that the DAO gene contains six exons and five introns. The gene encodes a polypeptide of 368 amino acids with a calculated molecular mass of 40,079 Da. Using an Escherichia coli protein expression system, the DAO protein of R. toruloides can easily be produced in an active form and purified in a large quantity.
Liao
G J
GJ
Food Industry Research and Development Institute, Taiwan, Republic of China.
Lee
Y J
YJ
Lee
Y H
YH
Chen
L L
LL
Chu
W S
WS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D005800:Genes, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000096:biosynthesis*; D010455:Peptides / Q000032:analysis; D014158:Transcription, Genetic; D015003:Yeasts / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D009841:Oligonucleotides; D010455:Peptides; D003605:D-Amino-Acid Oxidase